β1,3-galactosyltransferase and DNA encoding the enzyme

ABSTRACT

The present invention provides a protein having β1,3-galactosyltransferase activity, a DNA encoding the protein, a transformant comprising the DNA, a process for producing the protein using the transformant, and a process for producing a galactose-containing complex carbohydrate using the transformant.

This application is a national stage application of PCT/JP02/04034,filed Apr. 23, 2002, which itself claims benefit of Japanese patentapplication No. 123864, filed Apr. 23, 2001.

TECHNICAL FIELD

The present invention relates to a protein havingβ1,3-galactosyltransferase activity, a DNA encoding the protein, atransformant comprising the DNA, a process for producing a proteinhaving β1,3-galactosyltransferase activity using the transformant, and aprocess for producing a galactose-containing complex carbohydrate usingthe transformant.

BACKGROUND ART

β1,3-Galactosyltransferase genes, derived from mammals [JapanesePublished Unexamined Patent Application No. 181759/94; J. Biol. Chem.,272 24794 (1997); J. Biol. Chem., 273, 58 (1998); Eur. J. Biochem., 263,571 (1999); J. Neurosci. Res., 58, 318 (1999)] and derived from a bird[Acta Biochemica Polonica, 45, 451 (1998)] have been obtained. However,there is no example in which β1,3-galactosyltransferase gene derivedfrom an animal was expressed as an active protein in a microorganismsuch as Escherichia coli. Although there is an example in which the genederived from a bird was expressed in Escherichia coli [Acta BiochemicaPolonica, 45, 451 (1998)], its activity was very weak.

On the other hand, in the case of microorganisms, aβ1,3-galactosyltransferase gene has been obtained from Campylobacterjejuni and expressed in Escherichia coli as an active protein [J. BiolChem., 275, 3896 (2000); Mol. Microbiol., 37, 501 (2000)]. However, asthe substrate of the protein, the references describe onlyGalNAcβ1,4[NeuAcα2,3]Galβ1,4Glc-FCHASE in which its non-reducingterminal has N-acetylgalactosamine and its reducing terminal is labeledwith a fluorescent material [FCHASE(6-(5-fluoresceincarboxamido)hexanoic acid succimidyl ester)], and thereferences do not describe a complex carbohydrate having a modificationon its reducing terminal or a complex carbohydrate havingN-acetylglucosarnine on its non-reducing terminal.

Regarding Helicobacter pylori 26695, the full nucleotide sequence of itsgenomic DNA has been determined [Nature, 388, 539 (1997)], but a geneproduct which functions as a β1,3-galactosyltransferase has not beenknown. Furthermore, since a Galβ1,3GlcNAc structure in Helicobacterpylori disappeared by disruption of a gene which corresponds to HP0619gene of Helicobacter pylori 26695, HP0619 gene is expected to be aβ1,3-galactosyltransferase gene [Infect. Immun., 68, 5928 (2000)], butthere have been neither examples in which the gene was expressed in amicroorganism such as Escherichia coli nor descriptions regarding itsdetailed enzyme activity.

Among galactose-containing complex carbohydrates, complex carbohydrateshaving a Galβ1,3-R structure in the skeleton are particularly importantas synthesis intermediates of Lewis^(a) or Lewis^(b) which is a bloodgroup complex carbohydrate [J. Pediatr. Gastroenterol. Nutr., 30, 181(2000)], and is contained in human milk in a large amount and consideredto have a preventive effect against bacterial infection [Anal. Biochem.,2239, 218 (1994); J. Chromatogr., 685, 211 (1996); Lancet, 347, 1017(1996)].

For the production of the galactose-containing complex carbohydrateshaving a Galβ1,3-R structure, an extraction method from human milk[Carbohydr. Res., 178, 79 (1988)], a chemical synthesis method[Carbohydr. Res., 316, 121 (1999)] and the method using an enzyme[Glycoconjugate J., 16, 189 (1999)] have been reported, however, each ofthe methods has problems from the viewpoints of cost and productivity,and thus an industrial production method has not been established yet.

DISCLOSURE OF THE INVENTION

Objects of the present invention are to provide a protein havingβ1,3-galactosyltransferase activity, a DNA encoding the protein, atransformant comprising the DNA, a process for producing a proteinhaving β1,3-galactosyltransferase activity using the transformant, and aprocess for producing a galactose-containing complex carbohydrate usingthe transformant.

In order to solve the above problems, the present inventors haveconducted intensive studies and obtained a DNA corresponding to HP0619gene from Helicobacter pylori NCTC 11637 based on the sequenceinformation of Helicobacter pylori 26695 in which the sequence of thegenomic DNA was determined. As a result of the activity measurement ofthe gene product, the present inventors found that the gene product hasβ1,3-galactosyltransferase activity, and thus the present invention hasbeen completed.

Specifically, the present invention relates to the following (1) to(17):

-   (1) A protein comprising the amino acid sequence represented by SEQ    ID NO:1.-   (2) A protein which consists of an amino acid sequence in which at    least one amino acid is deleted, substituted, inserted or added in    the amino acid sequence represented by SEQ ID NO:1, and has    β1,3-galactosyltransferase activity.-   (3) A protein which has a homology of at least 80% with a protein    consisting of the amino acid sequence represented by SEQ ID NO:1,    and has β1,3 -galactosyltransferase activity.-   (4) A DNA encoding the protein according to any one of (1) to (3).-   (5) A DNA comprising the nucleotide sequence represented by SEQ ID    NO:2.-   (6) A DNA which hybridizes with a DNA consisting of the nucleotide    sequence represented by SEQ ID NO:2 under stringent conditions, and    encodes a protein having β1,3 -galactosyltransferase activity.-   (7) A recombinant DNA comprising the DNA according to any one of (4)    to (6).-   (8) A transformant comprising the recombinant DNA according to (7).-   (9) The transformant according to (8), which is obtained by using a    microorganism, a plant cell, an insect cell or an animal cell as a    host cell.-   (10) The transformant according to (9), wherein the microorganism    belongs to the genus Escherichia.-   (11) The transformant according to (10), wherein the microorganism    belonging to the genus Escherichia is Escherichia coli.-   (12) A process for producing a protein having    β1,3-galactosyltransferase activity, which comprises: culturing the    transformant according to any one of (8) to (11) in a medium to    produce and accumulate a protein having β1,3-galactosyltransferase    activity in the culture, and recovering the protein having    β1,3-galactosyltransferase activity from the culture.-   (13) A process for producing a galactose-containing complex    carbohydrate, which comprises: allowing a culture of the    transformant according to any one of (8) to (11) or a treated    product of the culture as an enzyme source,    uridine-5′-diphosphogalactose and an acceptor complex carbohydrate    to be present in an aqueous medium to produce and accumulate the    galactose-containing complex carbohydrate in the aqueous medium, and    recovering the galactose-containing complex carbohydrate from the    aqueous medium.-   (14) The process according to (13), wherein the treated product of    the culture is selected from the group consisting of a concentrated    product of the culture, a dried product of the culture, cells    obtained by centrifuging the culture, a dried product of the cells,    a freeze-dried product of the cells, a surfactant-treated product of    the cells, an ultrasonic-treated product of the cells, a    mechanically disrupted product of the cells, a solvent-treated    product of the cells, an enzyme-treated product of the cells, a    protein fraction of the cells, an immobilized product of the cells    and an enzyme preparation obtained by extracting from the cells.-   (15) The process according to (13), wherein the acceptor complex    carbohydrate is a complex carbohydrate comprising an oligosaccharide    having N-acetylglucosamine in its non-reducing terminal.-   (16) The process according to (15), wherein the oligosaccharide    having N-acetylglucosamine in its non-reducing terminal is selected    from the group consisting of GlcNAc, GlcNAcβ1,3Galβ1,4Glc and    GlcNAcβ1,3Galβ1,4GlcNAcβ1,3Galβ1,4Glc.-   (17) The process according to (13), wherein the galactose-containing    complex carbohydrate is selected from the group consisting of    lacto-N-biose (Galβ1,3GlcNAc), lacto-N-tetraose    (Galβ1,3GlcNAcβ1,3Galβ1,4Glc) and paralacto-N-hexaose    (Galβ1,3GlcNAcβ1,3Galβ1,4GlcNAcβ1,3Galβ1,4Glc).

The protein of the present invention is a protein having an activity oftransferring galactose to an acceptor complex carbohydrate havingN-acetylglucosamine in its non-reducing terminal via β1,3-linkage amongproteins having β1,3-galactosyltransferase activity. The origin of theprotein of the present invention is not limited, but is preferably aprotein derived from a microorganism, more preferably a protein derivedfrom a microorganism belonging to the genus Helicobacter, and mostpreferably a protein derived from a microorganism belonging toHelicobacter pylori. Specifically, the protein includes a proteincomprising the amino acid sequence represented by SEQ ID NO:1 and aprotein which consists of an amino acid sequence in which at least oneamino acid is deleted, substituted, inserted or added in the amino acidsequence represented by SEQ ID NO:1, and has β1,3 -galactosyltransferaseactivity.

The protein which consists of an amino acid sequence in which at leastone amino acid is deleted, substituted, inserted or added, and hasβ1,3-galactosyltransferase activity can readily be obtained by using amethod for introducing site-directed mutagenesis described in, forexample, Molecular Cloning, A Laboratory Manual, Second Edition, ColdSpring Harbor Laboratory Press (1989) (hereinafter referred to as“Molecular Cloning, Second Edition”); Current Protocols in MolecularBiology, John Wiley & Sons (1987-1997) (hereinafter referred to as“Current Protocols in Molecular Biology”); Nucleic Acids. Research, 10,6487 (1982); Proc. Natl. Acad. Sci. USA, 79 6409 (1982); Gene, 34, 315(1985); Nucleic Acids. Research, 13 4431 (1985); Proc. Natl. Acad. Sci.USA, 82, 488 (1985) and the like. For example, the protein can beobtained by introducing mutation(s) to DNA encoding a protein consistingof the amino acid sequence represented by SEQ ID NO:1.

The number of the amino acids which are deleted, substituted, insertedor added is not particularly limited; however, it is such a number thatdeletion, substitution, insertion or addition can be carried out by aknown method such as method for introducing site-directed mutation(s).The number is 1 to several tens, preferably 1 to 20, more preferably 1to 10, and most preferably 1 to 5.

The deletion, substitution, insertion or addition of at least one aminoacid residue in the amino acid sequence represented by SEQ ID NO:1 meansthat one or at least two amino acids are deleted, substituted, insertedor added at any position in the same sequence. The deletion,substitution, insertion or addition can be carried out in the same aminoacid sequence simultaneously. Also, the amino acid residue substituted,inserted or added can be natural or non-natural. The natural amino acidresidue includes L-alanine, L-asparagine, L-asparatic acid, L-glutamine,L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine,L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine,L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine, and thelike.

Herein, examples of amino acid residues which are substituted with eachother are shown below. Amino acid residues in the same group can readilybe substituted with each other.

Group A:

leucine, isoleucine, norleucine, valine, norvaline, alanine,2-aminobutanoic acid, methionine, O-methylserine, t-butylglycine,t-butylalanine, cyclohexylalanine;

Group B:

asparatic acid, glutamic acid, isoasparatic acid, isoglutamic acid,2-aminoadipic acid, 2-aminosuberic acid;

Group C:

asparagine, glutamine;

Group D:

lysine, arginine, ornithine, 2,4-diaminobutanoic acid,2,3-diaminopropionic acid;

Group E:

proline, 3-hydroxyproline, 4-hydroxyproline;

Group F:

serine, threonine, homoserine;

Group G:

phenylalanine, tyrosine.

Also, in order that the protein of the present invention hasβ1,3-galactosyltransferase activity, the protein has a homology ofpreferably at least 60% or more, more preferably 80% or more, and mostpreferably 95% or more, with the amino acid sequence represented by SEQID NO:1.

The identity of an amino acid sequence or a nucleotide sequence can bedetermined by using the algorithm BLAST by Karlin and Altschl [Proc.Natl. Acad. Sci. USA, 90,5873 (1993)] or FASTA [Methods Enzymol, 183 63(1990)]. The programs called BLASTN and BLASTX have developed based onthe above algorithm BLAST [J. Mol. Biol., 215, 403 (1990)]. In the caseof analyzing a nucleotide sequence by BLASTN based on BLAST, forexample, the parameter can be set to score=100, wordlength=12. Also, inthe case of analyzing an amino acid sequence by BLASTX based on BLAST,for example, the parameter can be set to score=50, wordlength=3. WhenBLAST and Gapped BLAST programs are used, a default parameter of eachprogram can be used. The specific analysis methods of using the aboveprograms are known (http://www.ncbi.nlm.nih.gov.).

Any DNA can be used as the DNA of the present invention, so long as itencodes the protein of the present invention. It is preferably a DNAderived from a microorganism, more preferably a DNA derived from amicroorganism belonging to the genus Helicobacter, and most preferably aDNA derived from Helicobacter pylori. Examples include:

-   (1) a DNA encoding a protein comprising the amino acid sequence    represented by SEQ ID NO:1,-   (2) a DNA comprising the nucleotide sequence represented by SEQ ID    NO:2,-   (3) a DNA encoding a protein which consists of an amino acid    sequence in which at least one amino acid is deleted, substituted,    inserted or added in the amino acid sequence represented by SEQ ID    NO:1, and has β1,3-galactosyltransferase activity, and-   (4) a DNA which hybridizes with the DNA according to any one of (1)    to (3) under stringent conditions and encodes a protein having    β1,3-galactosyltransferase activity.

The DNA which is hybridizable under stringent conditions is a DNAobtained by colony hybridization, plaque hybridization, Southernhybridization or the like using, as a probe, a part or a full length ofthe DNA according to any one of (1) to (3). Specifically, the DNAincludes a DNA which can be identified by carrying out hybridization at65° C. in the presence of 0.7-1.0 mol/l NaCl using a filter on which aDNA prepared from colonies or plaques is immobilized, and then washingthe filter with 0.1× to 2×SSC solution (the composition of 1×SSCsolution contains 150 mmol/l sodium chloride and 15 mmol/l sodiumcitrate) at 65° C. The hybridization can be carried out in accordancewith a known method described in, for example, Molecular Cloning, SecondEdition; Current Protocols in Molecular Biology; DNA Cloning 1: CoreTechniques, A Practical Approach, Second Edition, Oxford University(1995) or the like. Specifically, the DNA which is hybridizable includesa DNA having a homology of at least 60% or more, preferably 80% or more,and more preferably 95% or more, with the nucleotide sequencerepresented by SEQ ID NO:2 when calculated based on the above parametersusing above BLAST, FASTA or the like.

(1) Preparation of the DNA of the Present Invention

The full nucleotide sequence of the genomic DNA in Helicobacter pylori26695 was determined [Nature, 388, 539 (1997)], and the sequenceinformation of the gene of interest can be obtained by using the genomicDNA sequence database(http://www.ncbi.nlm.nih.gov/Entrez/Genome/org.html,http://www.tigr.org./tdb/).

The DNA encoding the protein of the present invention can be preparedfrom a microorganism belonging to the genus Helicobacter. Themicroorganism belonging to the genus Helicobacter includes Helicobacterpylori, and specifically Helicobacter pylori NCTC 11637 (ATCC 43504) andthe like.

The microorganism belonging to the Helicobacter pylori can be culturedby a known method [for example, Mol. Microbiol, 20, 833 (1996)].

After the culturing, a chromosomal DNA of the microorganism can beisolated and purified by a known method (for example, method describedin Current Protocols in Molecular Biology).

A DNA fragment containing the DNA of the present invention can beobtained by preparing a primer based on the nucleotide sequence of thegenome and then carrying out PCR [PCR Protocols, Academic Press (1990)]using the genomic DNA as a template.

Furthermore, the DNA of interest can be obtained according to ahybridization method by using the synthetic DNA designed based on thenucleotide sequence of the genome as a probe.

The nucleotide sequence of the DNA can be determined by inserting theobtained DNA as it is or after digestion with an appropriate restrictionenzyme, into a vector according to the usual method, and carrying outanalysis by the generally used nucleotide sequence analysis method suchas the dideoxy method [Proc. Natl. Acad. Sci. USA, 74 5463 (1977)] or amethod comprising the use of an apparatus for nucleotide sequenceanalysis such as 373A-DNA Sequencer (manufactured by Perkin-Elmer).

Based on the nucleotide sequence thus determined, the DNA of interestcan also be prepared by chemical synthesis using, for example, DNASynthesizer 8905 manufactured by Perceptive Biosystems or the like.

The thus obtained DNA includes a DNA comprising the sequence representedby SEQ ID NO:2.

The vector into which the DNA of the present invention is ligatedincludes pBluescript KS(+) (manufactured by Stratagene), pDIRECT[Nucleic Acids Res., 18 6069 (1990)], pCR-Script Amp SK(+) (manufacturedby Stratagene), pT7Blue manufactured by Novagen), pCR II (manufacturedby Invitrogen), pCR-TRAP (manufactured by Genehunter) and the like.

The microorganism containing the recombinant DNA comprising the DNAcomprising the sequence represented by SEQ ID NO:2 includes Escherichiacoli and the like.

Escherichia coli includes Escherichia coli XL 1-Blue, Escherichia coliXL2-Blue, Escherichia coli DH1, Escherichia coli MC1000, Escherichiacoli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichiacoli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichiacoli NY49, Escherichia coli MP347, Escherichia coli NM522, Escherichiacoli ME8415 and the like.

Any method can be used in the introduction method of the recombinantDNA, so long as it is a method for introducing DNA into the host cell.Examples include the method using a calcium ion [Proc. Natl. Acad. Sci.USA, 69, 2110 (1972)], the protoplast method (Japanese PublishedUnexamined Patent Application No. 248394/88), electroporation [NucleicAcid Res., 16, 6127 (1988)] and the like.

Escherichia coli containing the recombinant DNA comprising a DNAcomprising the nucleotide sequence represented by SEQ ID NO:2 includesEscherichia coli NM522/pGT116.

(2) Preparation of the Protein of the Present Invention.

The protein of the present invention can be produced by expressing theDNA of the present invention obtained by the method of (1) in a hostcell, for example, as shown below, by using a method described inMolecular Cloning, Second Edition, Current Protocols in MolecularBiology or the like.

Based on the DNA of the present invention, a DNA fragment of anappropriate length containing a portion which encodes the protein can beprepared, if necessary. In addition, productivity of the protein can beimproved by substituting a nucleotide in the nucleotide sequence of theprotein-coding region so that it has the most suitable codons for theexpression in the host.

A recombinant DNA is prepared by inserting the DNA into a downstream ofthe promoter of an appropriate expression vector.

A transformant which produces the protein used for the process of thepresent invention can be obtained by introducing the recombinant DNAinto a host cell suitable for the expression vector.

Any bacteria, yeasts, animal cells, insect cells, plant cells and thelike can be used as the host cell, so long as it can express the gene ofinterest.

The expression vectors include those which can replicate autonomously inthe above host cell or those which can be integrated into a chromosomeand have a promoter at such a position that the DNA of the presentinvention can be transcribed.

When a procaryote cell such as a bacterial cell is used as the hostcell, it is preferred that the recombinant DNA can replicateautonomously in the procaryote cell, and that the recombinant vectorcontains a promoter, a ribosome binding sequence, the DNA of the presentinvention and a transcription termination sequence. The vector mayfurther comprise a gene regulating the promoter.

The expression vector includes pHelix1 (manufactured by RocheDiagnostics), pKK233-2 (manufactured by Amersham Pharmacia Biotech),pSE280 (manufactured by Invitrogen), pGEMEX-1 (manufactured by Promega),pQE-8 (manufactured by QIAGEN), pET-3 (manufactured by Novagen), pKYP10(Japanese Published Unexamined Patent Application No. 110600/83),pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSA1 [Agric. Biol. Chem.,53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci. USA, 82, 4306 (1985)],pBluescript II SK(+), pBluescript II KS(−) (manufactured by Stratagene),pTrs30 [prepared from Escherichia coli JM109/pTrs30 (FERM BP-5407)],pTrs32 [prepared from Escherichia coli JM109/pTrs32 (FERM BP-5408)],pPAC31 (WO98/12343), pUC19 [Gene, 33, 103 (1985)], pSTV28 (manufacturedby Takara Shuzo), pUC118 (manufactured by Takara Shuzo), pPA1 (JapanesePublished Unexamined Patent Application No. 233798/88) and the like.

Any promoter can be used, so long as it can function in the host cell.Examples include promoters derived from Escherichia coli, phage and thelike, such as trp promoter (P_(trp)), lac promoter (P_(lac)), P_(L)promoter, P_(R) promoter and P_(SE) promoter, SPO1 promoter, SPO2promoter, penP promoter and the like. Also, artificially designed andmodified promoters, such as a promoter in which two P_(trp) are linkedin tandem, tac promoter, lacT7 promoter and letI promoter, can be used.

It is preferred to use a plasmid in which the space betweenShine-Dalgarno sequence, which is the ribosome binding sequence, and theinitiation codon is adjusted to an appropriate distance (for example, 6to 18 nucleotides).

The transcription termination sequence is not essential for therecombinant DNA of the present invention. However, it is preferred tolie a transcription terminating sequence immediately downstream of thestructural gene.

The procaryotes include microorganisms belonging to the generaEscherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium,Microbacterium, Pseudomonas and the like. Examples include Escherichiacoli XL1-Blue, Escherichia coli XL2-Blue, Escherichia coli DH1,Escherichia coli NM522, Escherichia coli MC1000, Escherichia coliKY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coliHB101, Escherichia coli No. 49, Escherichia coli W3 110, Escherichiacoli NY49, Serratia ficaria, Serratia fonticola, Serratia liquefaciens,Serratia marcescens, Bacillus subtilis, Bacillus amyloliquefaciens,Brevibacterium immariophilim ATCC 14068, Brevibacterium saccharolyticumATCC 14066, Corynebacterium ammoniagenes, Corynebacterium glutamicumATCC 13032, Corynebacterium glutamicum ATCC 14067, Corynebacteriumglutamicum ATCC 13869, Corynebacterium acetoacidophilum ATCC 13870,Microbacterium ammoniaphilum ATCC 15354, Pseudomonas sp. D-0110 and thelike.

Introduction of the recombinant DNA can be carried out by any methodsfor introducing DNA into the above-described host cells, such as themethod using a calcium ion [Proc. Natl. Acad. Sci. USA, 69, 2110(1972)], the protoplast method (Japanese Published Unexamined PatentApplication No. 248394/88) and electroporation [Nucleic Acids Res., 16,6127 (1988)].

When a yeast cell is used as the host cell, the expression vectorincludes YEp13 (ATCC 37115), YEp24 (ATCC 37051), YCp50 (ATCC 37419),pHS19, pHS15 and the like.

Any promoter can be used so long as it can function in yeast. Examplesinclude PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, gal 1promoter, gal 10 promoter, a heat shock polypeptide promoter, MFα1promoter, CUP 1 promoter and the like.

The host cell includes yeast strain belonging to the generaSaccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon,Schwanniomyces, Pichia, Candida and the like. Examples includeSaccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyceslactis, Trichosporon pullulans, Schwanniomyces alluvius, Pichiapastoris,Candida utilis and the like.

Introduction of the recombinant DNA can be carried out by any of themethods for introducing DNA into yeast, such as electroporation[Methods. Enzymol., 194, 182 (1990)], the spheroplast method [Proc.Natl. Acad. Sci. USA, 81, 4889 (1984)] and the lithium acetate method[J. Bacteriol., 153, 163 (1983)].

When an animal cell is used as the host cell, the expression vectorincludes pcDNAI and pcDM8 (manufactured by Funakoshi), pAGE107 (JapanesePublished Unexamined Patent Application No. 22979/91), pAS3-3 (JapanesePublished Unexamined Patent Application No. 227075/90), pCDM8 [Nature,329, 840 (1987)], pcDNAI/Amp (manufactured by Invitrogen), pREP4(manufactured by Invitrogen), pAGE103 [J. Biochem., 101, 1307 (1987)],pAGE210, pAMo, pAMoA and the like.

Any promoter can be used, so long as it can function in an animal cell.Examples include a promoter of IE (immediate early) gene ofcytomegalovirus (CMV), SV40 early promoter, a metallothionein promoter,a promoter of retrovirus, a heat shock promoter, SRα promoter and thelike. Also, the enhancer of the IE gene of human CMV can be usedtogether with the promoter.

The host cell includes mouse myeloma cell, rat myeloma cell, mousehybridoma cell, human Namalwa cell, Namalwa KJM-1 cell, human fetalkidney cell, human leukemia cell, African grivet kidney cell, Chinesehamster ovary (CHO) cell, HST5637 (Japanese Published Unexamined PatentApplication No. 299/88) and the like.

The mouse myeloma cell includes SP2/0, NS0 and the like. The rat myelomacell includes YB2/0 and the like. The human fetal kidney cell includesHEK293 (ATCC: CRL-1573) and the like. The human leukemia cell includesBALL-1 and the like. The African grivet kidney cell includes COS-1,COS-7 and the like.

Introduction of the recombinant DNA into animal cells can be carried outby any of methods for introducing DNA into animal cells, such aselectroporation [Cytotechnology, 3, 133 (1990)], the calcium phosphatemethod (Japanese Published Unexamined Patent Application No. 227075/90),the lipofection method [Proc. Natl. Acad Sci. USA, 84, 7413 (1987)], andthe method described in Virology, 52, 456 (1973).

When an insect cell is used as the host cell, the protein can beproduced by a known method described in, for example, BaculovirusExpression Vectors, A Laboratory Manual, W.H. Freeman and Company, NewYork (1992), Molecular Biology, A Laboratory Manual, Current Protocolsin Molecular Biology, Bio/Technology, 6, 47 (1988) or the like.

Specifically, a recombinant gene transfer vector and baculovirus areco-transfected into an insect cell to obtain a recombinant virus in asupernatant of the culture of its insect cell, and then an insect cellis infected with the resulting recombinant virus to produce the protein.

The transfer vector used in the method includes pVL1392, pVL1393 andpBlueBacIII (all manufactured by Invitrogen), and the like.

The baculovirus includes Autographa californica nuclear polyhedrosisvirus which infects insects of the family Barathra and the like.

The insect cell includes Spodoptera frugiperda ovary cell, Trichoplusiani ovary cell, silkworm ovary-derived culturing cell and the like.

Spodoptera frugiperda ovary cell includes Sf9 and Sf21 (BaculovirusExpression Vectors, A Laboratory Manual) and the like. Trichoplusia niovary cell includes High 5 and BTI-TN-5B1-4 (manufactured by Invitrogen)and the like. The cell line derived from silkworm ovary cell includesBombyx mori N4 and the like.

The method for co-transfecting the above transfer vector and the abovebaculovirus for the preparation of the recombinant virus includes thecalcium phosphate method (Japanese Published Unexamined PatentApplication No. 227075/90), the lipofection method [Proc. Natl. Acad.Sci. USA, 84, 7413 (1987)] and the like.

When a plant cell is used as the host cell, the expression vectorincludes Ti plasmid, a tobacco mosaic virus vector, and the like.

As the promoter, any promoter can be used, so long as it can function ina plant cell. Examples include 35S promoter of cauliflower mosaic virus(CaMV), rice actin 1 promoter and the like.

The host cell includes a plant cell and the like, such as tobacco,potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat and barley.

Introduction of the recombinant vector is carried out by the method forintroducing DNA into a plant cell, such as the Agrobacterium method(Japanese Published Unexamined Patent Application No. 140885/84,Japanese Published Unexamined Patent Application No. 70080/85, WO94/00977), electroporation (Japanese Published Unexamined PatentApplication No. 251887/85) and the method using a particle gun (JapanesePatent Nos. 2606856 and 2517813).

When expressed in yeast, an animal cell or an insect cell, aglycosylated or sugar chain-added protein can be obtained.

The protein of the present invention can be produced by culturing thetransformant of the present invention thus obtained in a medium toproduce and accumulate the protein in the culture, and recovering itfrom the culture.

Culturing of the transformant of the present invention in a medium iscarried out according to the conventional method as used in culturing ofthe host.

As a medium for culturing the transformant obtained by using, as thehost, prokaryote such as Escherichia coli, or eukaryote such as yeast,either a natural medium or a synthetic medium may be used, so long as itcontains a carbon source, a nitrogen source, an inorganic salt and thelike which can be assimilated by the organism and the transformant canbe cultured efficiently.

Any carbon source can be used, so long as the organism can assimilate,and it includes carbohydrates, such as glucose, fructose, sucrose,molasses containing them, starch and starch hydrolysate; organic acids,such as acetic acid and propionic acid; alcohols, such as ethanol andpropanol; and the like.

The nitrogen source includes ammonia, various ammonium salts ofinorganic acids or organic acids, such as ammonium chloride, ammoniumsulfate, ammonium acetate and ammonium phosphate; othernitrogen-containing compounds; peptone; meat extract; yeast extract;corn steep liquor; casein hydrolysate; soybean meal and soybean mealhydrolysate; various fermented cells and digested matter thereof; andthe like.

The inorganic salt includes potassium dihydrogen phosphate, dipotassiumhydrogen phosphate, magnesium phosphate, magnesium sulfate, sodiumchloride, ferrous sulfate, manganese sulfate, copper sulfate, calciumcarbonate and the like.

Culturing is usually carried out under aerobic conditions by shakingculture, submerged spinner culture under aeration or the like. Theculturing temperature is preferably from 15 to 40° C., and the culturingtime is generally from 5 hours to 7 days. The pH of the medium ispreferably maintained at 3.0 to 9.0 during the culturing. The pH can beadjusted using inorganic or organic acid, an alkali solution, urea,calcium carbonate, ammonia or the like.

Also, antibiotics, such as ampicillin and tetracycline, can be added tothe medium during culturing, if necessary.

When a microorganism transformed with an expression vector containing aninducible promoter is cultured, an inducer can be added to the medium,if necessary. For example, isopropyl-β-D-thiogalactopyranoside or thelike can be added to the medium when a microorganism transformed with anexpression vector containing lac promoter is cultured; or indoleacrylicacid or the like can be added thereto when a microorganism transformedwith an expression vector containing trp promoter is cultured.

The medium for culturing a transformant obtained using an animal cell asthe host includes generally used RPMI 1640 medium [The Journal of theAmerican Medical Association, 199, 519 (1967)], Eagle's MEM medium[Science, 122, 501 (1952)], DMEM medium [Virology, 8, 396 (1959)], and199 Medium [Proceeding of the Society for the Biological Medicine, 73, 1(1950)], as well as media to which fetal calf serum or the like has beenadded to the above media and the like.

Culturing is generally carried out at pH 6 to 8 and at 25 to 40° C. for1 to 7 days in the presence of 5% CO₂ or the like.

Furthermore, if necessary, antibiotics such as kanamycin, penicillin andstreptomycin, can be added to the medium during the culturing.

The medium for culturing a transformant obtained using an insect cell asthe host includes generally used TNM-FH medium (manufactured byPharmingen), Sf-900 II SFM (manufactured by Life Technologies), ExCell400 and ExCell 405 (both manufactured by JRH Biosciences), Grace'sInsect Medium [Nature, 195, 788 (1962)] and the like.

Culturing is generally carried out at pH 6 to 7 and at 25 to 30° C. for1 to 5 days or the like.

Furthermore, if necessary, antibiotics such as gentamicin can be addedto the medium during the culturing.

A transformant obtained by using a plant cell as the host cell can beused as the cell or after differentiating to a plant cell or organ. Themedium used in the culturing of the transformant includes Murashige andSkoog (MS) medium, White medium, media to which a plant hormone, such asauxin or cytokinine, has been added, and the like.

Culturing is carried out generally at a pH 5 to 9 and at 20 to 40° C.for 3 to 60 days.

Also, antibiotics, such as kanamycin and hygromycin, can be added to themedium during the culturing, if necessary.

As described above, the protein can be produced by culturing atransformant derived from a microorganism, animal cell or plant cellcontaining a recombinant DNA to which the DNA of the present inventionhas been inserted according to the general culturing method to therebyproduce and accumulate the protein, and recovering the protein from theculture.

The process for producing the protein of the present invention includesa method of intracellular expression in a host cell, a method ofextracellular secretion from a host cell, or a method of production onan outer membrane of the host cell, and depending on the methodselected, the host cell employed or the structure of the proteinproduced is suitably changed.

When the protein of the present invention is produced in a host cell oron an outer membrane of the host cell, the produced protein can beactively secreted extracellularly according to, for example, the methodof Paulson et al. [J. Biol. Chem., 264, 17619 (1989)], the method ofLowe et al. [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989); GenesDevelop., 4, 1288 (1990)], or the methods described in JapanesePublished Unexamined Patent Application No. 336963/93, WO94/23021, andthe like.

Specifically, the protein of the present invention can be activelysecreted extracellularly by producing it in the form that a signalpeptide has been added to the side of N-terminal of a protein containingan active site of the protein of the present invention according to therecombinant DNA technique.

Furthermore, the protein production can be increased utilizing a geneamplification system using a dihydrofolate reductase gene or the likeaccording to the method described in Japanese Published UnexaminedPatent Application No. 227075/90.

Moreover, the protein of the present invention can be produced byrediferentiating a gene-introduced animal or plant cell to develop agene-introduced transgenic animal (transgenic nonhuman animal) or plant(transgenic plant), and using the individual.

When the transformant is the animal individual or plant individual, theprotein can be produced by breeding or cultivating it to produce andaccumulate the protein, and recovering the protein from the animalindividual or plant individual.

The process for producing the protein of the present invention using theanimal individual includes a method for producing the protein of thepresent invention in a nonhuman animal developed by introducing a geneaccording to a known method [Am. J. Clin. Nutr., 63, 639S (1996), Am. J.Clin. Nutr.:, 63, 627S (1996), Bio/Technology, 9, 830 (1991)].

In the animal individual, the protein can be produced by breeding atransgenic nonhuman animal to which the DNA of the present invention hasbeen introduced to produce and accumulate the protein in the animal, andrecovering the protein from the animal. The protein produced in theanimal is accumulated in milk (Japanese Published Unexamined PatentApplication No. 309192/88), egg, and the like. Any promoter can be used,so long as it can function in the animal. Suitable examples include anα-casein promoter, a β-casein promoter, a β-lactoglobulin promoter, awhey acidic protein promoter, and the like, which are specific formammary glandular cells.

The process for producing the protein of the present invention using theplant individual includes a process for producing the protein bycultivating a transgenic plant to which the DNA encoding the protein ofthe present invention is introduced by a known method [Tissue Culture(Soshiki Baiyo), 20 (1994), Tissue Culture (Soshiki Baiyo), 21 (1995),Trends Biotechnol., 15, 45 (1997)] to produce and accumulate the proteinin the plant, and recovering the protein from the plant.

The protein produced by the transformant of the present invention can beisolated and purified by using the general method for isolating andpurifying an enzyme.

For example, when the protein of the present invention is produced as asoluble product in the host cells, the cells are collected bycentrifugation after culturing, suspended in an aqueous buffer, anddisrupted using an ultrasonicator, a French press, a Manton Gaulinhomogenizer, a Dynomill, or the like to obtain a cell-free extract.

From the supernatant obtained by centrifuging the cell-free extract, apurified product can be obtained by the general method used forisolating and purifying an enzyme, for example, solvent extraction,salting-out using ammonium sulfate or the like, desalting, precipitationusing an organic solvent, anion exchange chromatography using a resinsuch as diethylaminoethyl (DEAE)-Sepharose or DIAION HPA-75(manufactured by Mitsubishi Chemical), cation exchange chromatographyusing a resin such as S-Sepharose FF (manufactured by Pharmacia),hydrophobic chromatography using a resin such as butyl sepharose orphenyl sepharose, gel filtration using a molecular sieve, affinitychromatography, chromatofocusing, or electrophoresis such asisoelectronic focusing, alone or in combination thereof.

When the protein is produced as an inclusion body in the host cells, thecells are collected in the same manner, disrupted and centrifuged torecover the protein as the precipitate fraction, and then the inclusionbody of the protein is solubilized with a protein-denaturing agent.

The solubilized solution is diluted or dialyzed in a solution free froma protein denaturing agent or a solution having a diluted concentrationof a protein denaturing agent in such a degree that the protein is notdenatured to thereby constitute the normal tertiary structure of theprotein, and then a purified product of the protein can be obtained by apurification/isolation method similar to the above.

When the protein of the present invention or its glycosylated-derivativeis secreted out of cells, the protein or its derivative can be collectedin the culture supernatant.

Specifically, the culture medium is treated in a manner similar to theabove, such as centrifugation to obtain a solubilized fraction, fromwhich a purified product can be obtained using a purification/isolationmethod similar to the above.

The protein obtained by the above method includes a protein comprisingthe amino acid sequence represented by SEQ ID NO:1.

Furthermore, the protein of the present invention is produced as afusion protein with other protein, and can be purified using affinitychromatography using a substance having affinity to the fusion protein.For example, the polypeptide of the present invention is produced as afusion protein with protein A according to the method of Lowe et al.[Proc. Natl. Acad. Sci. USA, 86 8227 (1989); Genes Develop., 4, 1288(1990)], or the method described in Japanese Published Unexamined PatentApplication No. 336963/93 or WO94/23021, and the fusion protein can bepurified by affinity chromatography using immunoglubulin G.

Moreover, the protein of the present invention is produced as a fusionprotein with Flag peptide, and the fusion protein can be purified byaffinity chromatography using an anti-Flag antibody [Proc. Natl. Acad.Sci., USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)]. Inaddition, purification can be carried out by affinity chromatographyusing the antibody against the polypeptide per se.

Based on the amino acid sequence information of the protein thusobtained, the protein of the present invention can be produced by achemical synthesis method, such as Fmoc (fluorenylmethyloxycarbonyl)method or tBoc (t-butyloxycarbonyl) method. It can also be chemicallysynthesized using a peptide synthesizer manufactured by AdvancedChemTech, Perkin-Elmer, Pharmacia, Protein Technology Instrument,Synthecell-Vega, PerSeptive, Shimadzu Corporation, or the like.

(3) Preparation of Galactose-Containing Complex Carbohydrate

A galactose-containing complex carbohydrate can be produced in anaqueous medium by allowing a culture of the transformant obtained by theculturing described in (2) or a treated product of the culture as anenzyme source, uridine-5′-diphosphogalactose and an acceptor complexcarbohydrate to be present in the aqueous medium.

The treated product of culture includes a concentrated product of theculture, a dried product of the culture, cells obtained by centrifugingthe culture, a dried product of the cells, a freeze-dried product of thecells, a surfactant-treated product of the cells, an ultrasonic-treatedproduct of the cells, a mechanically disrupted product of the cells, asolvent-treated product of the cells, an enzyme-treated product of thecells, a protein fraction of the cells, an immobilized product of thecells, an enzyme preparation obtained by extracting from the cell, andthe like.

The enzyme source used in the production of a galactose-containingcomplex carbohydrate is used in a concentration of 1 mU/l to 1,000 U/l,preferably 10 mU/l to 100 U/l, when the activity capable of forming 1μmol of galactose-containing complex carbohydrate at 37° C. in 1 minuteis defined as 1 unit (U).

The uridine-5′-diphosphogalactose (hereinafter referred to as “UDP-Gal”)obtained by the above method includes purified or partly purifiedUDP-Gal, and UDP-Gal prepared in the aqueous medium using amicroorganism. That is, according to the method described in WO98/12343,using a culture of a microorganism capable of producing UTP from a UTPprecursor or a treated product of the culture, a culture of amicroorganism capable of producing UDP-Gal from UTP and galactose or atreated product of the culture as enzyme sources, and the enzymesources, a UTP precursor, a sugar and galactose are allowed to bepresent in the aqueous medium to thereby produce and accumulate UDP-Galin the aqueous medium.

Specifically, the UTP precursor includes orotic acid, the microorganismcapable of producing UTP includes Corynebacterium ammoniagenes, themicroorganism capable of producing UDP-Gal from UTP and galactoseincludes Escherichia coli NM522/pNT25/pNT32 (WO98/12343) containingplasmid pNT25 which expresses galT gene [Nucleic Acid Res., 14, 7705(1986)] and galK gene [Nucleic Acid Res., 13, 1841 (1985)] and plasmidpNT32 which expresses galU gene [J. Biochem., 115, 965 (1994)] and ppagene [J. Bacteriol., 170, 5901 (1988)].

The aqueous medium used in the production of a galactose-containingcomplex carbohydrate includes water; a buffer such as a phosphatebuffer, a carbonate buffer, an acetate buffer, a borate buffer, acitrate buffer and a tris buffer; alcohol, such as methanol and ethanol;ester such as ethyl acetate; ketone such as acetone; amide such asacetamide; and the like. Also, the culture of the microorganisms used asthe enzyme source can be used as an aqueous medium.

In producing a galactose-containing complex carbohydrate, a surfactantor an organic solvent may be added, if necessary. Any surfactant capableof accelerating the formation of a galactose-containing complexcarbohydrate can be used as the surfactant. Examples include nonionicsurfactants such as polyoxyethylene octadecylamine (e.g., Nymeen S-215,manufactured by Nippon Oil & Fats); cationic surfactants, such ascetyltrimethylammonium bromide and alkyldimethyl benzylammoniumchloride(e.g., Cation F2-40E, manufactured by Nippon Oil & Fats); anionicsurfactants such as lauroyl sarcosinate; tertiary amines such asalkyldimethylamine (e.g., Tertiary Amine FB, manufactured by Nippon Oil& Fats); and the like, which are used alone or as a mixture of two ormore. The surfactant is used generally in a concentration of 0.1 to 50g/l. The organic solvent includes xylene, toluene, fatty acid alcohol,acetone, ethyl acetate, and the like, which are used in a concentrationof generally 0.1 to 50 m/l.

The galactose-containing complex carbohydrate production reaction iscarried out in an aqueous medium having a pH 5 to 10, preferably pH 6 to8, at 20 to 50° C. for 1 to 96 hours. In the production reaction,inorganic salts, such as MnCl₂ and MgCl₂, can be added, if necessary.

The amount of the galactose-containing complex carbohydrate produced inthe aqueous medium can be determined, for example, using a carbohydrateanalysis system manufactured by Dionex [Anal. Biochem., 189:, 151(1990)] or the like.

The galactose-containing complex carbohydrate produced in the aqueousmedium can be recovered by the ordinary method using activated carbon,an ion exchange resin or the like.

BRIEF EXPLANATION OF THE DRAWING

FIG. 1 shows construction steps of 1,3-galactosyltransferase expressionplsmid pGT116.

Symbols in the drawing have the following meanings:

-   Amp^(r): ampicillin-resistant gene;-   P_(L): P_(L) promoter;-   cI857: temperature-sensitive repressor gene;-   HP0619: gene encoding β1,3-galactosyltransferase derived from    Helicobacter pylori.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is explained based on Examples, but the presentinvention is not limited to Examples.

EXAMPLE 1

Construction of a Strain in Which a Gene Derived from HelicobacterPylori is Expressed:

Helicobacter pylori NCTC 11637 was cultured by a known method [e.g.,Mol. Microbiol., 20, 833 (1996)].

After the culturing, a chromosomal DNA of the microorganism was isolatedand purified by a method described in Current Protocols in MolecularBiology.

Using the DNAs represented by SEQ ID NOs:3 and 4 which had beensynthesized using DNA synthesizer 8905 manufactured by PerceptiveBiosystems, a fragment containing a DNA corresponding to HP0619 gene ofHelicobacter pylori 26695 was amplified by the following method.

Using the above synthetic DNAs as primers, PCR was carried out by usingthe chromosomal DNA of Helicobacter pylori NCTC 11637 as a template. PCRwas carried out using 40 μl of a reaction solution containing 0.1 μg ofthe chromosomal DNA, 0.5 μmol/l of each primer, 2.5 units of Pfu DNApolymerase (manufactured by Stratagene), 4 μl of a 10× buffer for PfuDNA polymerase and 200 μmol/l of each deoxyNTP and repeating 30 times ofa reaction step consisting of 1 minute at 94° C., 2 minutes at 42° C.and 3 minutes at 72° C.

After confirming amplification of the fragment of interest by subjecting1/10 volume of the reaction solution to agarose gel electrophoresis, theremaining reaction solution was mixed with the same volume of TE [10mmol/l Tris-HCl, 1 mmol/l EDTA (pH 8.0)] saturated phenol/chloroform (1vol/1 vol).

After centrifugation of the mixed solution, two equivalents of coldethanol was added to the upper layer thus obtained, and the mixture wasallowed to stand at −80° C. for 30 minutes. By centrifuging thesolution, a DNA precipitate was obtained.

The DNA precipitate was dissolved in 20 μl of TE.

Using 5 μl of the dissolved solution, the DNA fragment was digested withrestriction enzymes ClaI and BamHI, the resulting DNA fragments wereseparated by agarose gel electrophoresis and then a DNA fragment of 1.4kb was recovered using GeneClean II Kit.

After 0.2 μg of pPAC31 DNA was digested with restriction enzymes ClaIand BamHI, the resulting DNA fragments were separated by agarose gelelectrophoresis and then a DNA fragment of 5.5 kb was recovered in thesame manner.

Using a ligation kit, the 1.4 kb and 5.5 kb fragments were subjected toa ligation reaction at 16° C. for 16 hours.

Using the ligation reaction solution, Escherichia coli NM522 wastransformed in accordance with the above known method, and thetransformants were spread on LB agar medium [10 g/l bacto tryptone(manufactured by Difco), 10 g/l yeast extract (manufactured by Difco), 5g/l sodium chloride, 15 g/l agarose] containing 50 mg/ml ampicillin andthen cultured overnight at 30° C.

By extracting a plasmid from the grown transformant colonies inaccordance with the above known method, an expression plasmid pGT116 wasobtained. The structure of the plasmid was confirmed by restrictionenzyme digestion (FIG. 1).

EXAMPLE 2

Production of lacto-N-biose (Galβ1,3GlcNAc):

Escherichia coli NM522/pGT116 obtained in Example 1 was inoculated intoa test tube charged with 8 ml of LB medium containing 50 μg/mlampicillin and cultured at 28° C. for 17 hours. The culture wasinoculated into a test tube charged with 8 ml of LB medium containing 50μg/ml ampicillin, with an inoculum size of 1%, and cultured at 28° C.for 4 hours and then at 40° C. for 3 hours. Wet cells were obtained bycentrifuging 0.1 ml of the culture. The wet cells could be stored at−20° C., if necessary, and it was able to use them by thawing prior touse.

The reaction was carried out at 37° C. for 24 hours in 0.1 ml of areaction solution composed of the thus obtained wet cells of Escherichiacoli NM522/pGT116, 50 mmol/l of a citrate buffer (pH 7.0), 10 mmol/lMnCl₂, 10 mmol/l GlcNAc, 10 mmol/l UDP-galactose and 4 g/l Nymeen S-215.

After completion of the reaction, the reaction product was analyzedusing a carbohydrate analysis system manufactured by Dionex (DX-500)under the following analyzing conditions to confirm that 0.6 mmol/l (221mg/l) lacto-N-biose was produced and accumulated in the reactionsolution.

Analyzing conditions:

-   Column: CarboPAC PA10-   Eluent: A; H₂O, B; 500 mmol/l NaOH-   Gradient: Concentration of the eluent B at 0 minute is adjusted to    8% and increased to 20% spending 21 minutes.-   Detector: Pulsed amperometry detector

EXAMPLE 3

Production of lacto-N-tetraose (Galβ1,3GlcNAcβ1,3Galβ1,4Glc) (1)

Escherichia coli NM522/pGT116 obtained in Example 1 was cultured by themethod described in Example 2. Wet cells were obtained by centrifuging0.1 ml of the culture. The wet cells could be stored at −20° C., ifnecessary, and it was able to use them by thawing prior to use.

The reaction was carried out at 37° C. for 24 hours in 0.1 ml of areaction solution composed of the obtained wet cells of Escherichia coliNM522/pGT116, 50 mmol/l of a citrate buffer (pH 7.0), 10 mmol/l MnCl₂,10 mmol/l GlcNAcβ1,3Galβ1,4Glc, 10 mmol/l UDP-galactose and 4 g/l NymeenS-215.

After completion of the reaction, the reaction product was analyzedusing a carbohydrate analysis system manufactured by Dionex (DX-500)under the analyzing conditions described in Example 2 to confirm that4.3 mmol/l (3.1 g/l) lacto-N-tetraose was produced and accumulated inthe reaction solution.

EXAMPLE 4

Production of para-lacto-N-hexaose

(Galβ1,3GlcNAcβ1,3Galβ1,4GlcNAcβ1,3Galβ1,4Glc):

Escherichia coli NM522/pGT116 obtained in Example 1 was cultured by themethod described in Example 2. Wet cells were obtained by centrifuging0.1 ml of the culture. The wet cells could be stored at −20° C., ifnecessary, and it was able to use them by thawing prior to use.

The reaction was carried out at 37° C. for 10 hours in 0.1 ml of areaction solution composed of the obtained wet cells of Escherichia coliNM522/pGT116, 50 mmol/l of a citrate buffer (pH 7.0), 10 mmol/l MnCl₂,10 mmol/l GlcNAcβ1,3Galβ1,4GlcNAcβ1,3Galβ1,4Glc, 10 mmol/l UDP-galactoseand 4 g/l Nymeen S-215.

After completion of the reaction, the reaction product was analyzedusing a carbohydrate analysis system manufactured by Dionex (DX-500)under the analyzing conditions described in Example 2 to confirm that2.2 mmol/l (2.4 g/l) p-lacto-N-hexaose was produced and accumulated inthe reaction solution.

EXAMPLE 5

Production of lacto-N-tetraose (Galβ1,3GlcNAcβ1,3Galβ1,4Glc) (2):

Escherichia coli NM522/pGT116 obtained in Example 1 was inoculated on125 ml of LB medium [10 g/l Bacto-Tryptone (manufactured by Difco), 10g/l yeast extract (manufactured by Difco), 5 g/l sodium chloride (pH7.3)] containing 50 μg/ml ampicillin in a one-liter capacitybaffle-equipped conical flask and cultured at 28° C. for 17 hours at 220rpm. After 125 ml of the culture was inoculated on 2.5 liters of TBmedium [10 g/l glucose, 12 g/l Bacto-Tryptone (manufactured by Difco),24 g/l yeast extract (manufactured by Difco), 2.3 g/l KH₂PO₄, 12.5 g/lK₂HPO₄ (pH not adjusted)] containing 50 μg/ml ampicillin in a five-litercapacity jar fermentor, and the mixture was cultured at 30° C. for 4hours and then at 40° C. for 3 hours at 600 rpm and 2.5 liter/minaeration. During the culturing, the pH of the medium was adjusted at 7.0using 28% aqueous ammonia, and glucose was added, if necessary. Wetcells were obtained by centrifuging the culture. The wet cells could bestored at −20° C., if necessary, and it was able to use them by thawingprior to use.

Escherichia coli NM522/pNT25/pNT32 obtained by a known method [Nat.Biotechnol., 16, 847 (1998)] was inoculated on 125 ml of LB mediumcontaining 50 μg/ml ampicillin and 10 μg/ml chloramphenicol in aone-liter capacity baffle-equipped conical flask and cultured at 28° C.for 17 hours at 220 rpm. After 125 ml of the culture was inoculated on2.5 liters of TB medium containing 50 μg/ml ampicillin and 10 μg/mlchloramphenicol in a five-liter capacity jar fermentor, the mixture wascultured at 30° C. for 4 hours and then at 40° C. for 3 hours at 600 rpmand 2.5 liter/min aeration. During the culturing, the pH of the mediumwas adjusted at 7.0 using 28% aqueous ammonia, and glucose was added, ifnecessary. Wet cells were obtained by centrifuging the culture. The wetcells could be stored at −20° C., if necessary, and it was able to usethem by thawing prior to use.

In a 300 ml capacity baffle-equipped conical flask, Corynebacteriumammoniagenes ATCC 21170 was inoculated into 25 ml of a liquid mediumcomprising a composition of 50 g/l glucose, 10 g/l polypeptone(manufactured by Nippon Pharmaceutical), 10 g/l yeast extract(manufactured by Oriental Yeast), 5 g/l urea, 5 g/l (NH₄)₂SO₄, 1 g/lKH₂PO₄, 3 g/l K₂HPO₄, 1 g/l MgSO₄.7H₂O, 0.1 g/l CaCl₂.2H₂O, 10 mg/lFeSO₄.7H₂O, 10 mg/l ZnSO₄.7H₂O, 20 mg/l MnSO₄.6H₂O, 20 mg/l L-cysteine,10 mg/l calcium D-pantothenate, 5 mg/l vitamin B1, 5 mg/l nicotinic acidand 30 μg/ml biotin (adjusted to pH 7.2 with NaOH), followed byculturing at 28° C. for 24 hours at 220 rpm.

In a two-liter capacity baffle-equipped conical flask, 20 ml of theculture was inoculated into 250 ml of a liquid medium having the samecomposition described above, the mixture was cultured at 28° C. for 24hours at 220 rpm. The thus obtained culture was used as a seed culturemedium.

In a five-liter capacity jar fermentor, 250 ml of the seed culturemedium was inoculated into 2.25 liters of a liquid medium comprising acomposition of 150 g/l glucose, 5 g/l meat extract (manufactured byKyokuto Pharmaceutical Industrial), 10 g/l KH₂PO₄, 10 g/l K₂HPO₄, 10 g/lMgSO₄.7H₂O, 0.1 g/l CaCl₂.2H₂O, 20 mg/l FeSO₄.7H₂O, 10 mg/l ZnSO₄.7H₂O,20 mg/l MnSO₄.4-6H₂O (separate sterilization), 15 mg/l β-alanine(separate sterilization), 20 mg/l L-cysteine, 5 mg/l vitamin B1(separate sterilization), 100 mg/ml biotin and 2 g/l urea (adjusted topH 7.2 with NaOH), the mixture was cultured at 32° C. for 24 hours at600 rpm and 2.5 liter/min aeration. During the culturing, the pH of themedium was adjusted at 6.8 using 28% aqueous ammonia.

Wet cells were obtained by centrifuging the culture. The wet cells couldbe stored at −20° C., if necessary, and it was able to use them bythawing prior to use.

After 30 ml of a reaction solution comprising a composition of 50 g/l ofwet cells of Escherichia coli NM522/pGT116, 50 g/l of wet cells ofEscherichia coli NM522/pNT25/pNT32, 150 g/l of wet cells ofCorynebacterium ammoniagenes ATCC 21170, 50 g/l galactose, 100 g/lGlcNAcβ1,3Galβ1,4Glc, 50 g/l fructose, 5 g/l orotic acid, 5 g/l KH₂PO₄,20 g/l K₂HPO₄, 5 g/l MgSO₄.7H₂O, 4 g/l Nymeen S-215 and 10 ml/l ofxylene was put into a 200 ml capacity beaker, and the reaction solutionwas stirred (900 rpm) using a magnetic stirrer to carry out the reactionat 32° C. for 24 hours. During the reaction, pH of the reaction solutionwas kept at 7.2 using 5 mol/l of NaOH, and fructose and KH₂PO₄ wereadded, if necessary.

After completion of the reaction, the reaction product was analyzed bythe method described in Example 2 to confirm that 61 g/llacto-N-tetraose was produced and accumulated in the reaction solution.

Industrial Applicability

According to the present invention, a protein havingβ1,3-galactosyltrasnferase activity can be produced in a large amount.Also, a galactose-containing complex carbohydrate can be producedefficiently by using the enzyme.

Free Text of Sequence Listing:

-   SEQ ID NO:3—Description of artificial sequence: Synthetic DNA-   SEQ ID NO:4—Description of artificial sequence: Synthetic DNA

1. An isolated DNA encoding a protein of (a) or (b): (a) a proteincomprising the amino acid sequence represented by SEQ ID NO:1; or (b) aprotein which has a homology of at least 95% with a protein consistingof the amino acid sequence represented by SEQ ID NO:1, and hasβ1,3-galactosyltransferase activity.
 2. An isolated DNA comprising thenucleotide sequence represented by SEQ ID NO:2.
 3. An isolated DNA whichhybridizes with a DNA consisting of the nucleotide sequence representedby SEQ ID NO:2 under stringent conditions, and encodes a protein havingβ1,3-galactosyltransferase acitivity, wherein the stringent conditionscomprise hybridization at 65° C. in the presence of 0.7-1.0 mol/l NaClusing a filter bearing immobilized DNA prepared from colonies orplaques, and then washing the filter with 0.1×SSC solution at 65° C. 4.A recombinant DNA comprising the DNA according to any one of claims 1 to3.
 5. A transformant comprising the recombinant DNA according to claim4.
 6. The transformant according to claim 5, which is obtained using amicroorganism, or an animal cell as a host cell.
 7. The transformantaccording to claim 6, wherein the host cell is a microorganism of thegenus Escherichia.
 8. The transformant according to claim 7, wherein themicroorganism is Escherichia coli.
 9. A process for producing a proteinhaving β1,3-galactosyltransferase activity, which comprises the stepsof: culturing the tranformant according to claim 5 in a medium, allowingthe cultured transformant to produce and accumulate a protein havingβ1,3-galactosyltransferase acitvity in the culture, and recovering theprotein having β1,3-galactosyltransferase activity from the culture.